Jun 09, 2023
Isolation and Processing of Embryonic and Postnatal Brains
- 1Northwestern University, Aligning Science Across Parkinson's (ASAP) Collaborative Research Network, Chevy Chase, MD 20815
Protocol Citation: Maryana Nissan, divya.darwinarulseeli 2023. Isolation and Processing of Embryonic and Postnatal Brains. protocols.io https://dx.doi.org/10.17504/protocols.io.e6nvwdpe9lmk/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: June 09, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 83162
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson's [ASAP-020600] through the Michael J. Fox Foundation for Parkinson's Research (MJFF)
Grant ID: ASAP-020600
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Abstract
This protocol describes:
-Harvesting embryos from females from embryonic day 10 [e10] to embryonic day 18 [e18]
-Brain isolation of embryonic (e10-e18) and postnatal brains (p0-p30)
-Processing and Freezing of embryonic and postnatal brains
Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
Harvest embryos from female mouse from embryonic day 10 (e10) to embryonic day 18 (e18)
Place pregnant female in an anesthetic chamber for 2 minutes
Euthanize female using cervical dislocation
Grip the abdominal wall with forceps and make incisions along the region to expose the abdomen
Cut away the uterus with scissors
Place the uterus in a petri dish filled with 1x phosphate-buffered solution [1x PBS]
Carefully poke the amniotic sac right next to the placenta to expose the embryo.
The embryo will slide out of the sac, do not handle the head of the embryo with the forceps
Behead at the neck of the embryo and collect a tail clip into its corresponding falcon. Each falcon should be filled with 4% paraformaldehyde [4% PFA]
Repeat to the remaining embryos in the uterus
Embryonic heads are left in 4% PFA overnight for fixation
Brain isolation of embryonic and postnatal brains
Brain isolation of embryonic and postnatal brains
Leave embryonic heads in 4% PFA for one night, and postnatal heads in 4% PFA for two nights
When fixation is complete, transfer the brain from 4% PFA into 1X-PBS
Place petri dish under a dissecting microscope [from Leica Microsystems]
Place head in the petri dish
The head is held down and stabilized by inserting the forceps into the eyes
Push the forceps inwards, making a medial cut
Using fine forceps, make horizontal cuts along the jaw. Do this to remove everything about the tongue, leaving behind the brain and skull only
Tilt the head on its side and use forceps to pull away between the skin and the skull to expose the brain. Do this while rotating the head as needed to remove the skin and skull
When the brain is exposed, slide forceps along the base of the skull and pinch off the brain to remove it from the head
Processing and Freezing of embryonic and postnatal brains
Processing and Freezing of embryonic and postnatal brains
When the brain is isolated, place the brain in 30% Sucrose prepared in 1X PBS
Wait until the brain sinks. For embryonic brains, this takes 3-16 hours while postnatal brains take 24-35 hours
When the brains have sunk, they are ready to be frozen
Fill a bucket with powdered dry ice
Place Isopentane bottle and the brain’s respective falcon on top of the dry ice. Make sure they are secured within the dry ice
Pour the brain into a petri dish
Gently scoop the brain from the petri dish and place it on top of a thickly folded paper towel
Roll a KimTech Dry wipe and use it to gently roll the brain along the paper towel. This will ensure that the brain is dry
Once the brain is dried, slowly drop the brain into the Isopentane solution
Leave embryonic brains for 15-20 minutes and postnatal brains for 25-30 minutes in the isopentane bottle for proper freezing
Once the freezing is done, gently scoop the brain out of the isopentane bottle and gently run the spoon over a KimTech dry wipe to dry the liquid. Make sure to work quickly in this step, so the brain does not thaw.
Transfer the brain into its respective falcon, and place the falcon in -80C.
Brains frozen with Isopentane can be stored in the -80C indefinitely.